(1) Summary of the Invention
The present invention relates to a process and bacterial cultures which are used to produce useful cyclic hydroxides, preferably aromatic monohydroxides or dihydroxides, from a compound having a benzene ring while inhibiting further degradation of the cyclic hydroxide. In particular, the present invention relates to a process using a phenol resistant Bacillus in a growth medium with an effective amount of tetracycline which inhibits the degradation of pyrocatechol as it is produced.
(2) Prior Art
Phenols present in the effluents of oil refineries, petrochemical plants and other industrial processes are hazardous pollutants and a continuing waste treatment concern. A number of mesophilic microorganisms have been reported to degrade phenol, including Pseudomonas, Alcaligenes, Streptomyces setonii, and the yeasts, such as Trichosporon cutaneum and Candida tropicalis (Neujahr, H. Y., and A. Gaal, Eur. J. Biochem. 35:386-400 (1973). In all cases, phenol proved growth inhibitory even at a modest concentration. It is likely that at least part of this growth inhibition is due to inhibition of phenol hydroxylase, which catalyses the first phenol degradation step. Although this enzyme has proven difficult to solubilize and characterize, inhibition of phenol hydroxylase activity in whole Pseudomonas cells at levels of 0.25 mM phenol is known. With T. cutaneum, where solubilization and purification of an NADPH-dependent phenol hydroxylase has been achieved, similar phenol substrate inhibition was observed and attributed to phenol interaction at sites other than the active site. Thus, although oxidation of phenol has been documented for many mesophilic microorganisms, growth is usually inhibited at low phenol concentrations, requiring low level feeding or slow release "phenol sinks" for successful utilization. This enzyme has been shown to be substrate inhibited above 0.3 mM phenol concentration both in Pseudomonas putida and Trichosporon yeast.
It is recognized that thermostable enzymes are frequently also more resistant to chemical denaturation. This suggests that if suitable pathways exist, biotransformation of environmental pollutants might be possible at concentrations toxic to mesophiles. Although little is known about aromatic pathways in thermophiles, Buswell, J. A., and D. G. Twomey, J. Gen. Microbiol. 87:377-379 (1975)) and Buswell, J. A., Biochem. and Biophysical Res. Comm. 60:934-941 (1974) isolated a strain of B. stearothermophilus capable of phenol degradation which utilized an NADH dependent phenol hydroxylase. This strain was inhibited by phenol concentrations above 5 mM, and phenol inhibition of the phenol hydroxylase was not studied.
U.S. Pat. Nos. 3,700,560 to Oppermann and 4,508,822 to Taylor describe the preparation of cyclic dihydroxy compounds from aromatic compounds using enzymes or bacteria. In general, the degradative pathway for the dicyclic dihydroxyl compounds is not present.
Pyrocatechol is an intermediate in the pathway for the degradation of phenol which is generally modified to a significant extent as it is formed. Because of its wide utilization in the plastic and photographic industries, efficient bioproduction of pyrocatechol could provide an alternative to chemical production processes if it could be achieved. Other cyclic hydroxides are also useful.